Identification of the CRP regulon using in vitro and in vivo transcriptional profiling - PubMed (original) (raw)
Identification of the CRP regulon using in vitro and in vivo transcriptional profiling
Dongling Zheng et al. Nucleic Acids Res. 2004.
Abstract
The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gene promoters. In this study, we have exploited a newly developed technique, run-off transcription/microarray analysis (ROMA) to define CRP-regulated promoters. Using ROMA, we identified 176 operons that were activated by CRP in vitro and 16 operons that were repressed. Using positive control mutants in different regions of CRP, we were able to classify the different promoters into class I or class II/III. A total of 104 operons were predicted to contain Class II CRP-binding sites. Sequence analysis of the operons that were repressed by CRP revealed different mechanisms for CRP inhibition. In contrast, the in vivo transcriptional profiles failed to identify most CRP-dependent regulation because of the complexity of the regulatory network. Analysis of these operons supports the hypothesis that CRP is not only a regulator of genes required for catabolism of sugars other than glucose, but also regulates the expression of a large number of other genes in E.coli. ROMA has revealed 152 hitherto unknown CRP regulons.
Figures
Figure 1
Logarithmic-scale scatter plots of spot intensities. The data were subjected to global and density-dependent normalization, and plotted on GeneSpring 4.2.1. Each data point corresponds to the average of two spots representing an individual MG1655 gene. The ratios were colour coded, with red for ratio above one and green for ratio below one. The middle line passes genes with no change at two conditions (ratio = 1) and the other two lines demarcate threshold values for genes with significant increase or decrease in transcription in response to CRP deletion (ratio = ±2). (a) Transcriptional profiles from reactions with wild-type CRP (wt. CRP) versus without CRP (−CRP). (b) Transcriptional profiles from reactions with AR1-mutated CRP (HL159) versus without CRP (−CRP). (c) Transcriptional profiles from reactions with AR2-mutated CRP (KE101) versus without CRP (−CRP).
Figure 2
Regulatory map of 50 highest CRP-regulated operons. The figure indicates the activation by HL159-CRP (middle) and KE101-CRP (right) relative to wild-type CRP (=1), generated by GeneSpring clustering in distance order, so that those with similar regulation pattern were grouped together. The relative activation changes are colour coded: yellow indicates that a gene is regulated at the same level as wild-type CRP, blue indicates that a gene is regulated at a much lower level than wild-type CRP and red indicates that a gene is regulated at a higher level.
Figure 3
Non-template strand sequences of promoters repressed by CRP. Except nirB and pncB, promoter regions are predicted by searching the −10 and −35 sequences upstream of 10 down-regulated genes with a CRP site. The predicted −10 and −35 hexamers are underlined and in boldface with the −10 regions aligned together. The potential CRP-binding sites are shown as shaded boxes. *ydfK and ynaE are both associated with different prophage (Quin and Rac, respectively) but have essentially identical sequences.
Figure 4
Venn diagram of the CRP regulon as identified by three genomic approaches: ROMA, in vivo transcriptional profiling, sequence search (5). The result for (a) 87 known (experimentally verified) CRP regulon collected in RegulonDB database and (b) all operons are shown. The number of operons identified by each approach is presented in a coloured circle. The numbers covered by more than one circles are operons identified by two or three methods.
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